Dexamethasone-mediated regulation of 3-methylcholanthrene-induced cytochrome P-450d mRNA accumulation in primary rat hepatocyte cultures.
We have previously demonstrated that cytochrome P-450c and P-450d mRNAs can be induced by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium (HDM) plus minimal salts (Silver, G., and Krauter, K. S. (1988) J. Biol. Chem. 263; 11802-11807). Such cultures were used to investigate the role of the individual hormonal components present in the medium in the hydrocarbon-mediated induction process. Replacing HDM with minimal salts plus 10% fetal bovine serum (FBS) resulted in a 4-fold reduction in the accumulation of P-450d mRNA in response to MCA. In contrast, no effect was seen on induced levels of P-450c mRNA. Mixing experiments, in which primary cultures of hepatocytes were grown in medium containing HDM plus 10% FBS, indicated that there was no negative acting component present in FBS, but rather there was a positive acting component present in the mixture of hormones in HDM which permitted P-450d induction by MCA. Testing the effects of singly deleting each of the 10 components in HDM on MCA-induced P-450d expression demonstrated that dexamethasone was the only factor which affected the induction of P-450d. Deletion of this component from HDM resulted in a 4-fold decrease in the maximum MCA induced expression of P-450d mRNA. Moreover, supplementation of minimal salts plus 10% FBS with dexamethasone restored full P-450d inducibility by MCA. Deletion of the other components from HDM had no effects on P-450d mRNA accumulation. Although substratum clearly contributed to the quality of primary hepatocyte cultures, we were unable to demonstrate any role of the substratum on MCA induction of P-450d. In vitro nuclear run-on experiments revealed that dexamethasone had little effect on the rate of transcription of the P-450d genes. Therefore, the effect of dexamethasone on induction must be at the posttranscriptional level.
Silver
G
G
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
Reid
L M
LM
Krauter
K S
KS
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D015342:DNA Probes; D003907:Dexamethasone / Q000494:pharmacology*; D006728:Hormones / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008748:Methylcholanthrene / Q000494:pharmacology*; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014158:Transcription, Genetic / Q000187:drug effects
D003470:Culture Media; D015342:DNA Probes; D006728:Hormones; D012333:RNA, Messenger; D008748:Methylcholanthrene; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System